Compounds and uses thereof for the modulation of hemoglobin

ABSTRACT

Provide herein are compounds and pharmaceutical compositions suitable as modulators of hemoglobin, methods and intermediates for their preparation, and methods for their use in treating disorders mediated by hemoglobin and disorders that would benefit from tissue and/or cellular oxygenation.

FIELD OF THE INVENTION

This invention provides compounds and pharmaceutical compositionssuitable as allosteric modulators of hemoglobin, methods andintermediates for their preparation, and methods for their use intreating disorders mediated by hemoglobin and disorders that wouldbenefit from tissue and/or cellular oxygenation.

STATE OF THE ART

Sickle cell disease is a disorder of the red blood cells, foundparticularly among those of African and Mediterranean descent. The basisfor sickle cell disease is found in sickle hemoglobin (HbS), whichcontains a point mutation relative to the prevalent peptide sequence ofhemoglobin (Hb).

Hemoglobin (Hb) transports oxygen molecules from the lungs to varioustissues and organs throughout the body. Hemoglobin binds and releasesoxygen through conformational changes. Sickle hemoglobin (HbS) containsa point mutation where glutamic acid is replaced with valine, allowingHbS to become susceptible to polymerization to give the HbS containingred blood cells their characteristic sickle shape. The sickled cells arealso more rigid than normal red blood cells, and their lack offlexibility can lead to blockage of blood vessels. U.S. Pat. No.7,160,910 discloses compounds that are allosteric modulators ofhemoglobin. However, a need exists for additional therapeutics that cantreat disorders that are mediated by Hb or by abnormal Hb such as HbS.

SUMMARY OF THE INVENTION

This invention relates generally to compounds and pharmaceuticalcompositions suitable as allosteric modulators of hemoglobin. In someaspects, this invention relates to methods for treating disordersmediated by hemoglobin and disorders that would benefit from tissueand/or cellular oxygenation.

In certain aspects of the invention, a compound of formula (I) isprovided:

or a tautomer thereof, or a pharmaceutically acceptable salt of eachthereof, wherein

-   ring A is an optionally substituted 4-10 membered cycloalkyl or 4-10    membered heterocycle containing up to 5 ring heteroatoms, wherein    the heteroatom is selected from the group consisting of O, N, S, and    oxidized forms of N and S;-   ring B is a C₆-C₁₀ aryl or 5-10 membered heteroaryl having 1-3    nitrogen atoms, preferably 1-2 nitrogen atoms and more preferably 1    nitrogen atom, or oxidized versions thereof, wherein the aryl or    heteroaryl is optionally substituted;-   is a single or a double bond;-   each Y and Z is independently CR¹⁰R¹¹, O, S, SO, SO₂, or NR¹²; each    R¹⁰ and R¹¹ independently is hydrogen or C₁-C₃ alkyl optionally    substituted with halo, OH, or C₁-C₆ alkoxy, or CR¹⁰R¹¹ is C═O; R¹²    is hydrogen or C₁-C₆ alkyl; provided that if one of Y and Z is O, S,    SO, SO₂, then the other is not CO, and provided that Y and Z are    both not heteroatoms or oxidized forms thereof;-   ring C is C₆-C₁₀ aryl;-   V¹ and V² independently are C₁-C₆ alkoxy; or V¹ and V² together with    the carbon atom they are attached to form a ring of formula:

-   wherein each V³ and V⁴ are independently O, S, or NH, provided that    when one of V³ and V⁴ is 5, the other is NH, and provided that V³    and V⁴ are both not NH; q is 1 or 2; each V₅ is independently C₁-C₆    alkyl or CO₂R⁶⁰, where each R⁶⁰ independently is C₁-C₆ alkyl or    hydrogen; t is 0, 1, 2, or 4; or CV¹V² is C≡V, wherein V is O,    NOR⁸⁰, or NNR⁸¹R⁸²;-   R⁵ is hydrogen, C₁-C₆ alkyl or a prodrug moiety R, wherein the C₁-C₆    alkyl is optionally substituted with 1-5 halo;-   R⁶ is halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylthio, C₁-C₆S(O)—,    C₁-C₆S(O)₂—, wherein the C₁-C₆ alkyl is optionally substituted with    1-5 halo; or-   R⁶ is 4-10 membered cycloalkyl or heterocycle substituted with an    R′R′N— moiety wherein each R′ is independently C₁-C₆ alkyl or    hydrogen;-   R⁸⁰ is optionally substituted C₁-C₆ alkyl;-   R⁸¹ and R⁸² independently are selected from the group consisting of    hydrogen, optionally substituted C₁-C₆ alkyl, COR⁸³, or CO₂R⁸⁴;-   R⁸³ is hydrogen or optionally substituted C₁-C₆ alkyl;-   R⁸⁴ is optionally substituted C₁-C₆ alkyl;-   k is 0 or 1; and-   p is 0, 1, 2 or 3.

In further aspects of the invention, a composition is providedcomprising any of the compounds described herein, and at least apharmaceutically acceptable excipient.

In still further aspects of the invention, a method is provided forincreasing oxygen affinity of hemoglobin S in a subject, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of any of the compounds or compositions describedherein.

In further aspects of the invention, a method is provided for treatingoxygen deficiency associated with sickle cell anemia, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of any of the compounds or compositions describedherein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asolvent” includes a plurality of such solvents.

As used herein, the term “comprising” or “comprises” is intended to meanthat the compositions and methods include the recited elements, but notexcluding others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the stated purpose. Thus,a composition or process consisting essentially of the elements asdefined herein would not exclude other materials or steps that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention. “Consisting of” shall mean excluding more than trace elementsof other ingredients and substantial method steps. Embodiments definedby each of these transition terms are within the scope of thisinvention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations. Each numerical parameter should atleast be construed in light of the number of reported significant digitsand by applying ordinary rounding techniques. The term “about” when usedbefore a numerical designation, e.g., temperature, time, amount, andconcentration, including range, indicates approximations which may varyby (+) or (−) 10%, 5% or 1%.

As used herein, C_(m)-C_(n), such as C₁-C₁₂, C₁-C₈, or C₁-C₆ when usedbefore a group refers to that group containing m to n carbon atoms.

The term “alkoxy” refers to —O-alkyl. The term alkylthio is —S-alkyl.

The term “alkyl” refers to monovalent saturated aliphatic hydrocarbylgroups having from 1 to 30 carbon atoms (i.e., C₁-C₃₀ alkyl) or 1 to 22carbon atoms (i.e., C₁-C₂₂ alkyl), 1 to 8 carbon atoms (i.e., C₁-C₈alkyl), or 1 to 4 carbon atoms. This term includes, by way of example,linear and branched hydrocarbyl groups such as methyl (CH₃—), ethyl(CH₃CH₂—), n-propyl (CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl(CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—), sec-butyl((CH₃)(CH₃—(CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl (CH₃CH₂CH₂CH₂CH₂—),and neopentyl (CH₃)₃CCH₂—).

The term “aryl” refers to a monovalent, aromatic mono- or bicyclic ringhaving 6-10 ring carbon atoms. Examples of aryl include phenyl andnaphthyl. The condensed ring may or may not be aromatic provided thatthe point of attachment is at an aromatic carbon atom. For example, andwithout limitation, the following is an aryl group:

The term “—CO₂H ester” refers to an ester formed between the —CO₂H groupand an alcohol, preferably an aliphatic alcohol. A preferred exampleincluded —CO₂R^(E), wherein R^(E) is alkyl or aryl group optionallysubstituted with an amino group.

The term “chiral moiety” refers to a moiety that is chiral. Such amoiety can possess one or more asymmetric centers. Preferably, thechiral moiety is enantiomerically enriched, and more preferably a singleenantiomer. Non limiting examples of chiral moieties include chiralcarboxylic acids, chiral amines, chiral amino acids, such as thenaturally occurring amino acids, chiral alcohols including chiralsteroids, and the likes.

The term “cycloalkyl” refers to a monovalent, preferably saturated,hydrocarbyl mono-, bi-, or tricyclic ring having 3-12 ring carbon atoms.While cycloalkyl, refers preferably to saturated hydrocarbyl rings, asused herein, it also includes rings containing 1-2 carbon-carbon doublebonds. Nonlimiting examples of cycloalkyl include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamentyl, and thelike. The condensed rings may or may not be non-aromatic hydrocarbylrings provided that the point of attachment is at a cycloalkyl carbonatom. For example, and without limitation, the following is a cycloalkylgroup:

The term “halo” refers to F, Cl, Br, and/or I.

The term “heteroaryl” refers to a monovalent, aromatic mono-, bi-, ortricyclic ring having 2-16 ring carbon atoms and 1-8 ring heteroatomsselected preferably from N, O, S, and P and oxidized forms of N, S, andP, provided that the ring contains at least 5 ring atoms. Nonlimitingexamples of heteroaryl include furan, imidazole, oxadiazole, oxazole,pyridine, quinoline, and the like. The condensed rings may or may not bea heteroatom containing aromatic ring provided that the point ofattachment is a heteroaryl atom. For example, and without limitation,the following is a heteroaryl group:

The term “heterocyclyl” or heterocycle refers to a non-aromatic, mono-,bi-, or tricyclic ring containing 2-12 ring carbon atoms and 1-8 ringheteroatoms selected preferably from N, O, S, and P and oxidized formsof N, S, and P, provided that the ring contains at least 3 ring atoms.While heterocyclyl preferably refers to saturated ring systems, it alsoincludes ring systems containing 1-3 double bonds, provided that thering is non-aromatic. Nonlimiting examples of heterocyclyl include,azalactones, oxazoline, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrofuranyl, and tetrahydropyranyl. The condensed rings may or maynot contain a non-aromatic heteroatom containing ring provided that thepoint of attachment is a heterocyclyl group. For example, and withoutlimitation, the following is a heterocyclyl group:

The term “hydrolyzing” refers to breaking an R^(H)—O—CO—, R^(H)—O—CS—,or an R^(H)—O—SO₂-moiety to an R^(H)—OH, preferably by adding wateracross the broken bond. A hydrolyzing is performed using various methodswell known to the skilled artisan, non limiting examples of whichinclude acidic and basic hydrolysis.

The term “oxo” refers to a C═O group, and to a substitution of 2 geminalhydrogen atoms with a C═O group.

The term “optionally substituted” refers to a substituted orunsubstituted group. The group may be substituted with one or moresubstituents; such as e.g., 1, 2, 3, 4 or 5 substituents. Preferably,the substituents are selected from the group consisting of oxo, halo,—CN, NO₂, —N₂+, —CO₂R¹⁰⁰, —OR¹⁰⁰, —SR¹⁰⁰, SOR¹⁰⁰, —SO₂R¹⁰⁰,—CONR¹⁰¹R¹⁰², —SO₂NR¹⁰¹R¹⁰², C₁-C₆ alkyl, C₁-C₆ alkoxy, —CR¹⁰⁰═C(R¹⁰⁰)₂,—CCR¹⁰⁰, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocyclyl, C₆-C₁₂ aryl and C₂-C₁₂heteroaryl, wherein each R¹⁰⁰ independently is hydrogen or C₁-C₈ alkyl;C₃-C₁₂ cycloalkyl; C₃-C₁₀ heterocyclyl; C₆-C₁₂ aryl; or C₂-C₁₂heteroaryl; wherein each alkyl, cycloalkyl, heterocyclyl, aryl, orheteroaryl is optionally substituted with 1-3 halo, 1-3 C₁-C₆ alkyl, 1-3C₁-C₆ haloalkyl or 1-3 C₁-C₆ alkoxy groups. Preferably, the substituentsare selected from the group consisting of chloro, fluoro, —OCH₃, methyl,ethyl, iso-propyl, cyclopropyl, vinyl, ethynyl, —CO₂H, —CO₂CH₃, —OCF₃,—CF₃ and —OCHF₂.

R¹⁰¹ and R¹⁰² independently is hydrogen; C₁-C₈ alkyl, optionallysubstituted with —CO₂H or an ester thereof, C₁-C₆ alkoxy, oxo,—CR¹⁰³═C(R¹⁰³)₂, —CCR, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocyclyl, C₆-C₁₂aryl, or C₂-C₁₂ heteroaryl, wherein each R¹⁰³ independently is hydrogenor C₁-C₈ alkyl; C₃-C₁₂ cycloalkyl; C₃-C₁₀ heterocyclyl; C₆-C₁₂ aryl; orC₂-C₁₂ heteroaryl; wherein each cycloalkyl, heterocyclyl, aryl, orheteroaryl is optionally substituted with 1-3 alkyl groups or 1-3 halogroups, or R¹⁰¹ and R¹⁰² together with the nitrogen atom they areattached to form a 5-7 membered heterocycle.

The term “pharmaceutically acceptable” refers to safe and non-toxic forin vivo, preferably, human administration.

The term “pharmaceutically acceptable salt” refers to a salt that ispharmaceutically acceptable.

The term “salt” refers to an ionic compound formed between an acid and abase. When the compound provided herein contains an acidicfunctionality, such salts include, without limitation, alkali metal,alkaline earth metal, and ammonium salts. As used herein, ammonium saltsinclude, salts containing protonated nitrogen bases and alkylatednitrogen bases. Exemplary, and non-limiting cations useful inpharmaceutically acceptable salts include Na, K, Rb, Cs, NH₄, Ca, Ba,imidazolium, and ammonium cations based on naturally occurring aminoacids. When the compounds utilized herein contain basic functionality,such salts include, without limitation, salts of organic acids, such ascarboxylic acids and sulfonic acids, and mineral acids, such as hydrogenhalides, sulfuric acid, phosphoric acid, and the likes. Exemplary andnon-limiting anions useful in pharmaceutically acceptable salts includeoxalate, maleate, acetate, propionate, succinate, tartrate, chloride,sulfate, bisalfate, mono-, di-, and tribasic phosphate, mesylate,tosylate, and the likes.

The terms “treat”, “treating” or “treatment”, as used herein, includealleviating, abating or ameliorating a disease or condition or one ormore symptoms thereof, preventing additional symptoms, ameliorating orpreventing the underlying metabolic causes of symptoms, inhibiting thedisease or condition, e.g., arresting or suppressing the development ofthe disease or condition, relieving the disease or condition, causingregression of the disease or condition, relieving a condition caused bythe disease or condition, or suppressing the symptoms of the disease orcondition, and are intended to include prophylaxis. The terms alsoinclude relieving the disease or conditions, e.g., causing theregression of clinical symptoms. The terms further include achieving atherapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication or amelioration of the underlying disorderbeing treated. Also, a therapeutic benefit is achieved with theeradication or amelioration of one or more of the physiological symptomsassociated with the underlying disorder such that an improvement isobserved in the individual, notwithstanding that the individual is stillbe afflicted with the underlying disorder. For prophylactic benefit, thecompositions are administered to an individual at risk of developing aparticular disease, or to an individual reporting one or more of thephysiological symptoms of a disease, even though a diagnosis of thisdisease has not been made.

The terms “preventing” or “prevention” refer to a reduction in risk ofacquiring a disease or disorder (i.e., causing at least one of theclinical symptoms of the disease not to develop in a subject that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease). The terms further include causing theclinical symptoms not to develop, for example in a subject at risk ofsuffering from such a disease or disorder, thereby substantiallyaverting onset of the disease or disorder.

The term “effective amount” refers to an amount that is effective forthe treatment of a condition or disorder by an intranasal administrationof a compound or composition described herein. In some embodiments, aneffective amount of any of the compositions or dosage forms describedherein is the amount used to treat a disorder mediated by hemoglobin ora disorder that would benefit from tissue and/or cellular oxygenation ofany of the compositions or dosage forms described herein to a subject inneed thereof.

The term “carrier” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells, e.g., red blood cells, or tissues.

As used herein, a “prodrug” is a compound that, after administration, ismetabolized or otherwise converted to an active or more active form withrespect to at least one property. To produce a prodrug, apharmaceutically active compound can be modified chemically to render itless active or inactive, but the chemical modification is such that anactive form of the compound is generated by metabolic or otherbiological processes. A prodrug may have, relative to the drug, alteredmetabolic stability or transport characteristics, fewer side effects orlower toxicity. For example, see the reference Nogrady, 1985, MedicinalChemistry A Biochemical Approach, Oxford University Press, New York,pages 388-392. Prodrugs can also be prepared using compounds that arenot drugs.

Compounds

In certain aspects of the invention, a compound of formula (I) isprovided:

or a tautomer thereof, or a pharmaceutically acceptable salt of eachthereof, wherein

-   ring A is an optionally substituted 4-10 membered cycloalkyl or 4-10    membered heterocycle containing up to 5 ring heteroatoms, wherein    the heteroatom is selected from the group consisting of O, N, S, and    oxidized forms of N and S;-   ring B is a C₆-C₁₀ aryl or 5-10 membered heteroaryl having 1-3    nitrogen atoms, preferably 1-2 nitrogen atoms and more preferably 1    nitrogen atom, or oxidized versions thereof, wherein the aryl or    heteroaryl is optionally substituted;-   is a single or a double bond;-   each Y and Z is independently CR¹⁰R¹¹, O, S, SO, SO₂, or NR¹²; each    R¹⁰ and R¹¹ independently is hydrogen or C₁-C₃ alkyl optionally    substituted with halo, OH, or C₁-C₆ alkoxy, or CR¹⁰R¹¹ is C═O; R¹²    is hydrogen or C₁-C₆ alkyl; provided that if one of Y and Z is O, S,    SO, SO₂, then the other is not CO, and provided that Y and Z are    both not heteroatoms or oxidized forms thereof;-   ring C is C₆-C₁₀ aryl;-   V¹ and V² independently are C₁-C₆ alkoxy; or V¹ and V² together with    the carbon atom they are attached to form a ring of formula:

-   wherein each V³ and V⁴ are independently O, S, or NH, provided that    when one of V³ and V⁴ is 5, the other is NH, and provided that V³    and V⁴ are both not NH; q is 1 or 2; each V₅ is independently C₁-C₆    alkyl or CO₂R⁶⁰, where each R⁶⁰ independently is C₁-C₆ alkyl or    hydrogen; t is 0, 1, 2, or 4; or CV¹V² is C═V, wherein V is O,    NOR⁸⁰, or NNR⁸¹R⁸²;-   R⁵ is hydrogen, C₁-C₆ alkyl or a prodrug moiety R, wherein the C₁-C₆    alkyl is optionally substituted with 1-5 halo;-   R⁶ is halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylthio, C₁-C₆S(O)—,    C₁-C₆S(O)₂—, wherein the C₁-C₆ alkyl is optionally substituted with    1-5 halo; or-   R⁶ is 4-10 membered cycloalkyl or heterocycle substituted with an    R′R′N-moiety wherein each R′ is independently C₁-C₆ alkyl or    hydrogen;-   R⁸⁰ is optionally substituted C₁-C₆ alkyl;-   R⁸¹ and R⁸² independently are selected from the group consisting of    hydrogen, optionally substituted C₁-C₆ alkyl, COR⁸³, or CO₂R⁸⁴;-   R⁸³ is hydrogen or optionally substituted C₁-C₆ alkyl;-   R⁸⁴ is optionally substituted C₁-C₆ alkyl;-   k is 0 or 1; and-   p is 0, 1, 2 or 3.

In certain embodiments, t is 0. In certain embodiments, t is 1. Incertain embodiments, t is 2. In certain embodiments, t is 3.

Preferably, in certain embodiments, Y and Z are both not a heteroatom ora heteroatom containing moiety. Preferably, one of Y and Z is amethylene or substituted methylene and the other is a heteroatom or aheteroatom containing moiety. More preferably, Y is an alkylene, and Zis a heteroatom or a heteroatom containing moiety, which, yet morepreferably is oxygen.

Preferably, V¹ and V² together with the carbon atom they are attached toform a ring of formula:

In some embodiments, V¹ and V² independently are C₁-C₆ alkoxy; or V¹ andV² together with the carbon atom they are attached to form a ring offormula:

wherein each V³ and V⁴ are independently O, S, or NH, provided that whenone or V³ and V⁴ is S the other is NH, and provided that V³ and V⁴ areboth not NH; q is 1 or 2; each V₅ is independently C₁-C₆ alkyl orCO₂R⁶⁰, where each R⁶⁰ independently is C₁-C₆ alkyl or hydrogen; t is 0,1, 2, or 4; or CV¹V² is C═V, wherein V is O, and wherein the remainingvariables are defined herein.

In certain aspects of the invention, the compound of Formula (I) is ofFormula (II):

wherein the remaining variables are defined herein.

In certain aspects of the invention, the compound of Formula (I) is ofFormula (IIA):

wherein

the variables are defined herein.

In some embodiments, ring A is optionally substituted with 1-3: halo,C₁-C₆ alkyl, COR¹⁵ and/or COOR¹⁵; wherein R¹⁵ is optionally substitutedC₁-C₆ alkyl, optionally substituted C₆-C₁₀ aryl, optionally substituted5-10 membered heteroaryl containing up to 5 ring heteroatoms, oroptionally substituted 4-10 membered heterocycle containing up to 5 ringheteroatoms, wherein the heteroatom is selected from the groupconsisting of O, N, S, and oxidized forms of N and S.

In some embodiments, ring B is optionally substituted with 1-3: halo,C₁-C₆ alkyl COR¹⁵ and/or COOR¹⁵; wherein R¹⁵ is optionally substitutedC₁-C₆ alkyl, optionally substituted C₆-C₁₀ aryl, optionally substituted5-10 membered heteroaryl containing up to 5 ring heteroatoms, oroptionally substituted 4-10 membered heterocycle containing up to 5 ringheteroatoms, wherein the heteroatom is selected from the groupconsisting of O, N, S, and oxidized forms of N and S.

In some embodiments, the compound is selected from the group consistingof

or an N oxide thereof, wherein

R¹⁴ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, COR¹⁵ or COOR¹⁵;

R¹⁵ is optionally substituted C₁-C₆ alkyl, optionally substituted C₈-C₁₀aryl, optionally substituted 5-10 membered heteroaryl containing up to 5ring heteroatoms, or optionally substituted 4-10 membered heterocyclecontaining up to 5 ring heteroatoms, wherein the heteroatom is selectedfrom the group consisting of O, N, S, and oxidized forms of N and S;

x is 0, 1, or 2;

p is 0, 1, and 2; and

m is 0, 1 or 2.

In one embodiment, the compound is

Prodrug Moiety

In one aspect, R is hydrogen, a phosphate or a diphosphate containingmoiety, or another promoiety or prodrug moiety. Preferably the prodrugmoiety imparts at least a 2 fold, more preferably a 4 fold, enhancedsolubility and/or bioavailability to the active moiety (where R ishydrogen), and more preferably is hydrolyzed in vivo. The promoietiesare structurally and functionally defined herein.

In one embodiments, R is —COR⁹⁰, CO₂R⁹¹, or CONR⁹²R⁹³ wherein

R⁹⁰ and R⁹¹ independently are C₁-C₆ alkyl, C₃-C₈ cycloalkyl, 4-9membered heterocycle, or a 5-10 membered heteroaryl, each containing atleast 1 basic nitrogen moiety; and

R⁹² and R⁹³ independently are C₁-C₆ alkyl; C₃-C₈ cycloalkyl, 4-9membered heterocycle, or a 5-10 membered heteroaryl, each containing atleast 1 basic nitrogen moiety; or R⁹² and R⁹³ together with the nitrogenatom they are bonded to for a 4-9 member heterocycle substituted with atleast 1 amino, C₁-C₆ alkyl amino, or di C₁-C₆ alkylamino group.

In certain embodiments, R is —C(O)R³¹, C(O)OR³¹, or CON(R¹³)₂,

each R³¹ is independently a C₁-C₆ alkyl; C₃-C₈ cycloalkyl, 4-9 memberedheterocycle, or a 5-10 membered heteroaryl, containing at least 1 basicnitrogen moiety; and

each R¹³ independently are C₁-C₆ alkyl; C₃-C₈ cycloalkyl, 4-9 memberedheterocycle, or a 5-10 membered heteroaryl, containing at least 1 basicnitrogen moiety; or 2 R¹³ together with the nitrogen atom they arebonded to for a 4-9 member heterocycle substituted with at least 1amino, C₁-C₆ alkyl amino, or di C₁-C₆ alkylamino group.

Preferably, R¹ is isopropyl.

In one aspect, R is C(O)OR³¹, C(S)OR³¹, C(O)SR³¹ or COR³¹, wherein R³¹is as defined herein.

In one embodiment, R³¹ is a group of the formula (CR³²R³³)_(e)NR³⁴R³⁵,wherein

each R³² and R³³ is independently H, a C₁-C₈ alkyl, C₃-C₉ heterocyclyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, C₃-C₉ heteroaryl or R³² and R³³ togetherwith the carbon atom they are bond to form a C₃-C₈ cycloalkyl, C₆-C₁₀aryl, C₃-C₉ heterocyclyl or C₃-C₈ heteroaryl ring system, or 2 adjacentR³² moieties or 2 adjacent R³³ moieties together with the carbon atomthey are bond to form a C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, C₃-C₉heterocyclyl or C₃-C₈ heteroaryl ring system;

each R³⁴ and R³⁵ is a C₁-C₈ alkyl, C₃-C₉ heterocyclyl, C₃-C₈ cycloalkyl,or R³⁴ and R³⁵ together with the nitrogen atom they are bond to form aC₃-C₈ cycloalkyl or C₃-C₉ heterocyclyl ring system;

each heterocyclic and heteroaryl ring system is optionally substitutedwith C₁-C₃ alkyl, —OH, amino and carboxyl groups; and

e is an integer of from 1 to 4.

In some less preferred embodiments R³⁴ and R³⁵ can be hydrogen.

In one embodiment, the subscript e is preferably 2 and each R³² and R³³is preferably independently selected from the group, H, CH₃, and amember in which R³² and R³³ are joined together to form a cyclopropyl,cyclobutyl, cyclopentyl; cyclohexyl, or1,1-dioxo-hexahydro-lΔ⁶-thiopyran-4-yl or tetrahydropyran-4-yl group.

With regard to the prodrug group, preferred embodiments are compoundswherein NR³⁴R³⁵ is morpholino.

In one embodiment, R is:

wherein

each R³² and R³³ is independently H, C₁-C₈ alkyl, or optionally, if bothpresent on the same substituent, may be joined together to form a C₃-C₈cycloalkyl, C₆-C₁₀ aryl, C₃-C₉ heterocyclyl or C₃-C₉ heteroaryl ringsystem.

Within this embodiment, each R³² and R³³ is independently, H, CH₃, orare joined together to form a cyclopropyl, cyclopbutyl, cyclopentyl,cyclohexyl, 1,1-dioxo-hexahydro-lλ⁶-thiopyran-4-yl ortetrahydropyran-4-yl group.

In a preferred embodiment, linkage of the prodrug moiety to the rest ofthe active molecule is stable enough so that the serum half life of theprodrug is from about 8 to about 24 hours.

In an embodiment of the invention, the prodrug moiety comprises atertiary amine having a pKa near the physiological pH of 7.5. Any amineshaving a pKa within 1 unit of 7.5 are suitable alternatives amines forthis purpose. The amine may be provided by the amine of a morpholinogroup. This pKa range of 6.5 to 8.5 allows for significantconcentrations of the basic neutral amine to be present in the mildlyalkaline small intestine. The basic, neutral form of the amine prodrugis lipophilic and is absorbed through the wall of the small intestineinto the blood. Following absorption into the bloodstream, the prodrugmoiety is cleaved by esterases which are naturally present in the serumto release an active compound.

Examples of R include, without limitation:

In another embodiment, R is as tabulated below:

R R¹ m R³⁴ R³⁵ NR³⁴R³⁵ C(O)(CH₂)_(m)NR ³⁴ R ³⁵ isopropyl 2 Me MeC(O)(CH₂)_(m)NR ³⁴ R ³⁵ isopropyl 3 Me Me C(O)(CH₂)_(m)NR ³⁴ R ³⁵isopropyl 4 Me Me C(O)(CH₂)_(m)NR ³⁴ R ³⁵ isopropyl 2

C(O)(CH₂)_(m)NR ³⁴ R ³⁵ isopropyl 3

C(O)(CH₂)_(m)NR ³⁴ R ³⁵ isopropyl 4

C(O)O(CH₂)_(m)NR³⁴R³⁵ isopropyl 2 Me Me C(O)O(CH₂)_(m)NR³⁴R³⁵ isopropyl3 Me Me C(O)O(CH₂)_(m)NR³⁴R³⁵ isopropyl 4 Me Me C(O)O(CH₂)_(m)NR³⁴R³⁵isopropyl 2

C(O)O(CH₂)_(m)NR³⁴R³⁵ isopropyl 3

C(O)O(CH₂)_(m)NR³⁴R³⁵ isopropyl 4

P(O)(OH)₂ isopropylan N oxide thereof, or a pharmaceutically acceptable salt of eachthereof.

In another aspect, R is,

wherein

R³⁶ is lower alkyl (e.g. C₁-C₆ alkyl).

In yet another aspect, R is:

wherein X¹, V¹ and X² are as defined herein.

In one embodiment, X¹ is selected from the group consisting of O, S andNR³⁷ wherein R³⁷ is hydrogen or C₁-C₆ alkyl;

Y¹ is —C(R³⁸)₂ or a sugar moiety, wherein each R³⁸ is independentlyhydrogen or C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₉ heterocyclyl, C₆-C₁₀aryl, or C₃-C₉ heteroaryl;

X² is selected from the group consisting of halogen, C₁-C₆ alkoxy,diacylglycerol, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆alkylthio, a PEG moiety, a bile acid moiety, a sugar moiety, an aminoacid moiety, a di- or tri-peptide, a PEG carboxylic acid, and —U—Vwherein

U is O or S; and

V is selected from the group consisting of C₁-C₆ alkyl, C₃-C₈cycloalkyl, C₃-C₉ heterocyclyl, C₆-C₁₀ aryl, C₃-C₉ heteroaryl, C(W²)X³,PO(X³)₂, and SO₂X³;

wherein W² is O or NR³⁹

wherein R³⁹ is hydrogen or C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₉hetrocyclyl, C₆-C₁₀ aryl, or C₃-C₉ heteroaryl; and

each X³ is independently amino, hydroxyl, mercapto, C₁-C₆ alkyl,heteroalkyl, cycloalkyl, hetrocyclyl, aryl, or heteroaryl, C₁-C₆ alkoxy,C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ alkylthio, a bile acid basedalkoxy group, a sugar moiety, a PEG moiety, and —O—CH₂—CH(OR⁴⁰)CH₂X⁴R⁴⁰,

wherein:

X⁴ is selected from the group consisting of O, S, S═O, and SO₂; and

each R⁴⁰ is independently C₁₀-C₂₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₉heterocyclyl, C₆-C₁₀ aryl, or C₃-C₉ heteroaryl, C₁-C₈ alkylene, or C₁-C₈heteroalkylene.

Each heterocyclic and heteroaryl ring system is optionally substitutedwith C₁-C₃ alkyl, —OH, amino and carboxyl groups.

In one embodiment, the present invention utilizes the following Y¹groups: CH₂, CHMe, CH(isopropyl), CH(tertiarybutyl), C(Me)₂, C(Et)₂,C(isopropyl)₂, and C(propyl)₂.

In another embodiment, the present invention utilizes the following X²groups:

—OMe, —OEt, —O-isopropyl, O-isobutyl, O-tertiarybutyl, —O—COMe,—O—C(═O)(isopropyl), —O—C(O)(isobutyl), —O—C(═O)(tertiarybutyl),—O—C(═O)—NMe₂, —O—C(═O)—NHMe, —O—C(═O)—NH₂, —O—C(═O)—N(H)—CH(R⁴¹)—CO₂Etwherein R⁴¹ is a side chain C₁-C₆ alkyl, or C₃-C₉ heterocyclyl groupselected from the side chain groups present in essential amino acids;—O—P(═O)(OMe)₂, —O—P(═O)(O-isopropyl)₂, and —O—P(═O)(O-isobutyl)₂. Eachheterocyclic is optionally substituted with one or more, preferably,1-3, C₁-C₃ alkyl, —OH, amino and/or carboxyl groups.

In another embodiment, In one embodiment, R is:

wherein

X³ is independently C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₉ heterocyclyl,C₆-C₁₀ aryl, or C₃-C₉ heteroaryl; and

R⁴² is independently hydrogen or C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₉heterocyclyl, C₆-C₁₀ aryl, or C₃-C₉ heteroaryl.

Each heterocyclic is optionally substituted with one or more,preferably, 1-3, C₁-C₃ alkyl, —OH, amino and/or carboxyl groups.

In one embodiment, R is:

wherein

each X³ is independently amino, hydroxyl, mercapto, C₁-C₆ alkyl, C₃-C₈cycloalkyl, C₃-C₉ heterocyclyl, C₆-C₁₀ aryl, or C₃-C₉ heteroaryl, C₁-C₆alkoxy, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, C₁-C₆ alkylthio, a bileacid based alkoxy group, a sugar moiety, a PEG moiety, and—O—CH₂—CH(OR⁴⁰)CH₂X⁴R⁴⁰,

-   -   wherein:

X⁴ is selected from the group consisting of O, S, S═O, and SO₂; and

each R⁴⁰ is independently C₁₀-C₂₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₉heterocyclyl, C₆-C₁₀ aryl, C₃-C₉ heteroaryl, C₁-C₈ alkylene, or C₁-C₈heteroalkylene; and

R⁴² is independently hydrogen or C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₉heterocyclyl, C₆-C₁₀ aryl, or C₃-C₉ heteroaryl.

In some embodiments, R⁴² is independently hydrogen or C₁-C₆ alkyl, C₃-C₈cycloalkyl, C₃-C₈ heterocyclyl, C₆-C₁₀ aryl, or C₃-C₈ heteroaryl; andeach X³ independently is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₉heterocyclyl, C₆-C₁₀ aryl, or C₃-C₉ heteroaryl, C₁-C₆ alkoxy, C₁-C₆alkylamino, C₁-C₆ dialkylamino, or C₁-C₆ alkylthio.

In some embodiments, R is represented by the following structures:

wherein, in the above examples, R⁴³ is C₁₀-C₂₂ alkyl or alkylene, R⁴⁴ isH or C₁-C₆ alkyl and R⁴⁵ represents side chain alkyl groups present innaturally occurring alpha amino acids;

wherein R⁴⁶ is (CH₂)_(n), f=2-4, and CO—R⁴⁷—NH₂ represents an aminoacylgroup; or

wherein R⁴⁶ is (CH₂)_(n) n=2-4, R⁴⁷ is (CH₂)_(n), n=1-3 and R⁴⁹ is O orNMe.

In one embodiment, R is:

In one aspect, R is —C(R²⁰⁰R²⁰¹)O(R²⁰²R²⁰³)P(O)OR²⁰⁴NR²⁰⁵R²⁰⁶, whereineach R²⁰⁰, R²⁰¹, R²⁰², R²⁰³, R²⁰⁴R²⁰⁵ and R²⁰⁶ is independently H, aC₁-C₈ alkyl, C₃-C₉ heterocyclyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, C₃-C₉heteroaryl, wherein each alkyl, heterocyclyl, cycloalkyl, aryl, andheteroaryl is optionally substituted.

In some embodiments, R is —CH(R²⁰¹)OCH₂P(O)OR²⁰⁴NHR²⁰⁶, wherein R²⁰¹ isC₁-C₈ alkyl, R²⁰⁴ is phenyl, optionally substituted. In one embodiment,R²⁰⁶ is —CHR²⁰⁷C(O)OR²⁰⁸ wherein R²⁰⁷ is selected from the groupconsisting of the naturally occurring amino acid side chains and CO₂Hesters thereof and R²⁰⁸ is C₁-C₈ alkyl. In one embodiment, R²⁰⁶ is C₁-C₆alkyl, optionally substituted with 1-3, CO₂H, SH, NH₂, C₆-C₁₀ aryl, andC₂-C₁₀ heteroaryl.

In some embodiments, R is:

In one embodiment, R is:

wherein Y¹ is —C(R³⁸)₂, wherein each R³⁸ is independently hydrogen orC₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₉ heterocyclyl, C₆-C₁₀ aryl, or C₃-C₉heteroaryl.

Various polyethylene glycol (PEG) moieties and synthetic methods relatedto them that can be used or adapted to make compounds of the inventionare described in U.S. Pat. Nos. 6,608,076; 6,395,266; 6,194,580;6,153,655; 6,127,355; 6,111,107; 5,965,566; 5,880,131; 5,840,900;6,011,042 and 5,681,567.

In one embodiment, R is

wherein

R⁵⁰ is —OH or hydrogen;

R⁵¹ is —OH, or hydrogen;

W is —CH(CH₃)W¹;

wherein W¹ is a substituted C₁-C₈ alkyl group containing a moiety whichis optionally negatively charged at physiological pH,

said moiety is selected from the group consisting of CO₂H, SO₃H, SO₂H,—P(O)(OR⁵²)(OH), —OP(O)(OR⁵²)(OH), and OSO₃H,

wherein R⁵² is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₈ heterocyclyl, C₆-C₁₀aryl, or C₃-C₈ heteroaryl.

Each heterocyclic and heteroaryl ring system is optionally substitutedwith one or more, preferably 1-3, C₁-C₃ alkyl, —OH, amino and/orcarboxyl groups.

In one embodiment, R is:

wherein R⁵³ is H or C₁-C₆ alkyl.

In another aspect, R is SO₃H.

In another aspect, R comprises a cleavable linker, wherein the term“cleavable linker” refers to a linker which has a short half life invivo. The breakdown of the linker Z in a compound releases or generatesthe active compound. In one embodiment, the cleavable linker has a halflife of less than ten hours. In one embodiment, the cleavable linker hasa half life of less than an hour. In one embodiment, the half life ofthe cleavable linker is between one and fifteen minutes. In oneembodiment, the cleavable linker has at least one connection with thestructure: C*—C(═X*)X*—C* wherein C* is a substituted or unsubstitutedmethylene group, and X* is S or O. In one embodiment, the cleavablelinker has at least one C*—C(═O)O—C* connection. In one embodiment, thecleavable linker has at least one C*—C(═O)S—C* connection. In oneembodiment, the cleavable linker has at least one—C(═O)N*—C*—SO₂—N*-connection, wherein N* is —NH— or C₁-C₆ alkylamino.In one embodiment, the cleavable linker is hydrolyzed by an esteraseenzyme.

In one embodiment, the linker is a self-immolating linker, such as thatdisclosed in U.S. patent publication 2002/0147138, to Firestone; PCTAppl. No. US05/08161 and PCT Pub. No. 2004/087075. In anotherembodiment, the linker is a substrate for enzymes. See generallyRooseboom et al., 2004, Pharmacol. Rev. 56:53-102.

Pharmaceutical Compositions

In further aspects of the invention, a composition is providedcomprising any of the compounds described herein, and at least apharmaceutically acceptable excipient.

In another aspect, this invention provides a composition comprising anyof the compounds described herein, and a pharmaceutically acceptableexcipient.

Such compositions can be formulated for different routes ofadministration. Although compositions suitable for oral delivery willprobably be used most frequently, other routes that may be used includetransdermal, intravenous, intraarterial, pulmonary, rectal, nasal,vaginal, lingual, intramuscular, intraperitoneal, intracutaneous,intracranial, and subcutaneous routes. Suitable dosage forms foradministering any of the compounds described herein include tablets,capsules, pills, powders, aerosols, suppositories, parenterals, and oralliquids, including suspensions, solutions and emulsions. Sustainedrelease dosage forms may also be used, for example, in a transdermalpatch form. All dosage forms may be prepared using methods that arestandard in the art (see e.g., Remington's Pharmaceutical Sciences,16^(th) ed., A. Oslo editor, Easton Pa. 1980).

Pharmaceutically acceptable excipients are non-toxic, aidadministration, and do not adversely affect the therapeutic benefit ofthe compound of this invention. Such excipients may be any solid,liquid, semi-solid or, in the case of an aerosol composition, gaseousexcipient that is generally available to one of skill in the art.Pharmaceutical compositions in accordance with the invention areprepared by conventional means using methods known in the art.

The compositions disclosed herein may be used in conjunction with any ofthe vehicles and excipients commonly employed in pharmaceuticalpreparations, e.g., talc, gum arabic, lactose, starch, magnesiumstearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffinderivatives, glycols, etc. Coloring and flavoring agents may also beadded to preparations, particularly to those for oral administration.Solutions can be prepared using water or physiologically compatibleorganic solvents such as ethanol, 1,2-propylene glycol, polyglycols,dimethylsulfoxide, fatty alcohols, triglycerides, partial esters ofglycerin and the like.

Solid pharmaceutical excipients include starch, cellulose, hydroxypropylcellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk and the like. Liquid andsemisolid excipients may be selected from glycerol, propylene glycol,water, ethanol and various oils, including those of petroleum, animal,vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineraloil, sesame oil, etc. In certain embodiments, the compositions providedherein comprises one or more of α-tocopherol, gum arabic, and/orhydroxypropyl cellulose.

In one embodiment, this invention provides sustained releaseformulations such as drug depots or patches comprising an effectiveamount of a compound provided herein. In another embodiment, the patchfurther comprises gum Arabic or hydroxypropyl cellulose separately or incombination, in the presence of alpha-tocopherol. Preferably, thehydroxypropyl cellulose has an average MW of from 10,000 to 100,000. Ina more preferred embodiment, the hydroxypropyl cellulose has an averageMW of from 5,000 to 50,000.

Compounds and pharmaceutical compositions of this invention maybe usedalone or in combination with other compounds. When administered withanother agent, the co-administration can be in any manner in which thepharmacological effects of both are manifest in the patient at the sametime. Thus, co-administration does not require that a singlepharmaceutical composition, the same dosage form, or even the same routeof administration be used for administration of both the compound ofthis invention and the other agent or that the two agents beadministered at precisely the same time. However, co-administration willbe accomplished most conveniently by the same dosage form and the sameroute of administration, at substantially the same time. Obviously, suchadministration most advantageously proceeds by delivering both activeingredients simultaneously in a novel pharmaceutical composition inaccordance with the present invention.

Methods of Treatment

In aspects of the invention, a method is provided for increasing tissueand/or cellular oxygenation, the method comprising administering to asubject in need thereof a therapeutically effective amount of any of thecompounds or compositions described herein.

In aspects of the invention, a method is provided for increasing oxygenaffinity of hemoglobin S in a subject, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of any of the compounds or compositions described herein.

In aspects of the invention, a method is provided for treating acondition associated with oxygen deficiency, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of any of the compounds or compositions described herein.

In further aspects of the invention, a method is provided for treatingoxygen deficiency associated with sickle cell anemia, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of any of the compounds or compositions describedherein.

In further aspects of the invention, a method is provided for treatingsickle cell disease, the method comprising administering to a subject inneed thereof a therapeutically effective amount of a compound of any ofthe compounds or compositions described herein. In still further aspectsof the invention, a method is provided for treating cancer, a pulmonarydisorder, stroke, high altitude sickness, an ulcer, a pressure sore,Alzheimer's disease, acute respiratory disease syndrome, and a wound,the method comprising administering to a subject in need thereof atherapeutically effective amount of a compound of any of the compoundsor compositions described herein.

Synthetic Methods

Certain methods for making the compounds described herein are alsoprovided. The reactions are preferably carried out in a suitable inertsolvent that will be apparent to the skilled artisan upon reading thisdisclosure, for a sufficient period of time to ensure substantialcompletion of the reaction as observed by thin layer chromatography,¹H-NMR, etc. If needed to speed up the reaction, the reaction mixturecan be heated, as is well known to the skilled artisan. The final andthe intermediate compounds are purified, if necessary, by various artknown methods such as crystallization, precipitation, columnchromatography, and the likes, as will be apparent to the skilledartisan upon reading this disclosure.

An illustrative and non-limiting method for synthesizing a compound offormula (I), is schematically shown below.

In the following Schemes,

refer to rings A, B and C as described herein.

X and X⁵ each represent a leaving group and are independently selectedfrom Cl, F, Br, and I.

X⁶ represents CHR¹⁴, NR¹⁴, O, S(O)_(x); wherein x is 0, 1, or 2;

Y⁵ represents a leaving group selected from Cl, F, Br, I, OSO₂R¹⁷ andOSO₂Ar;

R¹⁷ is C₁-C₆ alkyl;

n is 0, 1, or 2;

Ar is phenyl optionally substituted with 1-3 halo or C₁-C₄ alkyl groups.

General Synthetic Schemes

General Method A for Preparing Aryloxy Ether Analogs (4a) fromSubstituted Methylene Alcohol (1) and Hydroxyl Aryl Aldehyde Derivative(3a).

A hydroxyl arylaldehyde derivative (3a) (0.1-2 mmol) mixture withsubstituted methylene alcohol (1) (0.8 to 1.2 eq) and PPh₃ (1-1.5 eq) inanhydrous THF (1-10 ml) was stirred under nitrogen until completedissolution. The solution was cooled to 0° C. on ice bath and DIAD orDEAD (1.1 eq) in THF or toluene was added dropwise over a 1-20 minperiod. The ice cooling bath was allowed to expire over 90 min and themixture was stirred at RT for 2-48 hours. The mixture was stirred for 10min, then filtered through a pad of silica. The silica was washed withethyl acetate 2-20 mL. The combined filtrates were evaporated and theresidue was dried on highvac. The residue was purified by preparativeHPLC or flash silica gel chromatography.

General Method B for Preparing Aryloxy Ether Analogs (4a) fromSubstituted Methylene Halide (2) and Hydroxyl Aryl Aldehyde Derivatives(3a).

A mixture of hydroxyl arylaldehyde derivatives (3a) (0.1-2 mmol, 1-4eq.), substituted methylene chloride or bromide (2) (1 eq), and K₂CO₃(2-5 eq.) (catalytic amount of NaI or Bu₄NI may also be added) in DMF oracetonitrile (1 to 10 mL) was stirred at RT or heating up to 120° C. for0.5-8 h under nitrogen atmosphere. In workup A, water was added to thereaction mixture, the precipitated product was collected, washed withwater, and then subjected to preparative HPLC or flash silica gelchromatography purification. In workup B (for products that did notprecipitate), diluted HCl or aqueous NH₄Cl was added at 0° C. toadjusted the pH to ˜7, the reaction mixture was partitioned betweenethyl acetate or dichloromethane and aqueous sodium chloride and theorganic layer separated, dried, and solvent removed under vacuum toafford crude product which was purified by automated silica gel columnchromatography using appropriate solvents mixture (e.g., ethylacetate/hexanes).

General Method C for Preparing Substituted Methylene Chloride (2a).

To a solution of substituted methylene alcohol (1) (0.1 to 2 mmol) inDCM (1-10 mL) was added SOCl₂ dropwise (2 eq to 5 eq) at 0° C. or RT.The reaction mixture was stirred at RT for 10 min to 6 h, or untilreaction is judged complete (LC/MS). The reaction mixture isconcentrated to dryness over a rotavap. The crude chloride residue wassuspended in toluene, sonicated and concentrated to dryness. The processwas repeated three times and dried under vacuum to give the substitutedmethylene chloride (2), usually as an off-white solid, which was usedfor next step without further purification. Alternatively, a solution ofaqueous 1N Na₂CO₃ is then added to produce a solution of pH˜8. themixture was extracted with DCM (3×10-50 mL), dried over sodium sulfate,and concentrated to the crude substituted methylene chloride (2a), whichis then purified by column chromatography on silica gel (0-100% ethylacetate-hexanes).

General Method D for Preparing Substituted Methylene Bromide (2b).

To a solution of substituted methylene alcohol (1) (0.1 to 2 mmol) inDCM (1-10 mL) was added Ph₃PBr₂ dropwise (2 eq to 5 eq) at 0° C. or RT.The reaction mixture was stirred at RT for 10 min to 2 h, or untilreaction is judged complete (LC/MS). The reaction mixture isconcentrated to dryness over a rotavap. The residue purified by columnchromatography on silica gel (0-100% ethyl acetate-hexanes) to affordthe pure bromide 2b.

Similarly, N-linked heterocyclic analogs (compound 5) can also besynthesized from amination procedures developed by Buchwald and Hartwig.

Protected amides of formula —CONHR⁹⁵ and —CONHOR⁹⁵ can be convertede.g., hydrolyzed to the corresponding amides according to methods knownto the skilled artisan.

Prodrug Synthesis

Syntheses of the ester prodrugs start with the free carboxylic acidbearing the tertiary amine. The free acid is activated for esterformation in an aprotic solvent and then reacted with a free alcoholgroup in the presence of an inert base, such as triethyl amine, toprovide the ester prodrug. Activating conditions for the carboxylic acidinclude forming the acid chloride using oxalyl chloride or thionylchloride in an aprotic solvent, optionally with a catalytic amount ofdimethyl formamide, followed by evaporation. Examples of aproticsolvents, include, but are not limited to methylene chloride,tetrahydrofuran, and the like. Alternatively, activations can beperformed in situ by using reagents such as BOP(benzotriazol-1-yloxytris(dimethylamino) phosphoniumhexafluorophosphate, and the like (see Nagy et al., 1993, Proc. Natl.Acad. Sci. USA 90:6373-6376) followed by reaction with the free alcohol.Isolation of the ester products can be affected by extraction with anorganic solvent, such as ethyl acetate or methylene chloride, against amildly acidic aqueous solution; followed by base treatment of the acidicaqueous phase so as to render it basic; followed by extraction with anorganic solvent, for example ethyl acetate or methylene chloride;evaporation of the organic solvent layer; and recrystallization from asolvent, such as ethanol. Optionally, the solvent can be acidified withan acid, such as HCl or acetic acid to provide a pharmaceuticallyacceptable salt thereof. Alternatively the crude reaction can be passedover an ion exchange column bearing sulfonic acid groups in theprotonated form, washed with deionized water, and eluted with aqueousammonia; followed by evaporation.

Suitable free acids bearing the tertiary amine are commerciallyavailable, such as 2-(N-morpholino)-propionic acid,N,N-dimethyl-beta-alanine, and the like. Non-commercial acids can besynthesized in straightforward manner via standard literatureprocedures.

Carbonate and carbamate prodrugs can be prepared in an analogous way.For example, amino alcohols and diamines can be activated usingactivating agents such as phosgene or carbonyl diimidazole, to providean activated carbonates, which in turn can react with the alcohol and/orthe phenolic hydroxy group on the compounds utilized herein to providecarbonate and carbamate prodrugs.

Various protecting groups and synthetic methods related to them that canbe used or adapted to make compounds of the invention can be adaptedfrom the references Testa et al., Hydrolysis in Drug and ProdrugMetabolism, June 2003, Wiley-VCH, Zurich, 419-534 and Beaumont et al.,Curr. Drug Metab. 2003, 4:461-85.

Provided herein is a method of synthesizing an acyloxymethyl version ofa prodrug by adapting a method from the reference Sobolev et al., 2002,J. Org. Chem. 67:401-410.

R⁵¹ is C₁-C₆ alkyl.

Provided herein is a method for synthesizing a phosphonooxymethylversion of a prodrug by adapting a method from Mantyla et al., 2004, J.Med. Chem. 47:188-195.

Provided herein is a method of synthesizing an alkyloxymethyl version ofa prodrug

R⁵² is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₃-C₉ heterocyclyl, C₆-C₁₀ aryl,or C₃-C₉ heteroaryl.

Examples

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

In the examples below as well as throughout the application, thefollowing abbreviations have the following meanings. If not defined, theterms have their generally accepted meanings.

-   -   ° C.=degrees Celsius    -   RT=Room temperature    -   min=minute(s)    -   h=hour(s)    -   μL=Microliter    -   mL=Milliliter    -   mmol=Millimole    -   eq=Equivalent    -   mg=Milligram    -   MS=Mass spectrometry    -   LC-MS=Liquid chromatography-mass spectrometry    -   HPLC=High performance liquid chromatography    -   NMR=Nuclear magnetic resonance    -   EtOAc=Ethyl acetate    -   Ph₃PBr₂=Triphenylphosphine dibromide    -   DMF=N,N-Dimethylformamide    -   DCM=Dichloromethane    -   DMSO=Dimethyl sulfoxide    -   THF=Tetrahydrofuran    -   DIAD=Diisopropyl azodicarboxylate    -   DEAD=Diethyl azodicarboxylate

Preparation of2-[[2-[(3R)-3-fluoropyrrolidin-1-yl]pyridin-3-yl]methoxy]-6-hydroxybenzaldehyde

Step 1: (R)-ethyl 2-(3-fluoropyrrolidin-1-yl)nicotinate

To a solution of ethyl 2-fluoronicotinate (0.074 g, 0.48 mmol) in DMF(0.3 mL) was added diisopropylethyl amine (0.25 mL, 1.4 mmol), and(R)-3-fluoropyrrolidine (0.090 g, 0.72 mmol). The resulting mixture wasirradiated with microwaves (100° C.) for 1 h and loaded directly onto asilica column. Eluting the column with EtOAc/hexanes (0-100%) provided(R)-ethyl 2-(3-fluoropyrrolidin-1-yl)nicotinate as a clear oil (0.100 g,94% yield); MS (ESI) m/z 239 [M+H]⁺.

Step 2: (R)-(2-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)methanol

To a cooled (0° C.) solution of (R)-methyl2-(3-fluoropyrrolidin-1-yl)nicotinate in THF (5 mL) was added a solutionof lithium aluminum hydride (1M in THF). The reaction mixture wasstirred for 1 h and then 20 μL of H₂O was added followed by 20 μL of 15%NaOH (aq) and then 60 μL of additional water. The slurry was stirred for1 h and filtered and the resulting residue was washed with ether. Thecombined organic layers were dried over MgSO₄ and concentrated in vacuo.Purification by column chromotography (EtOAc/hexanes, 0-100%) provided(R)-(2-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)methanol (0.081 g, 92%yield). MS (ESI) m/z 197 [M+H]⁺.

Step 3: (R)-3-(chloromethyl)-2-(3-fluoropyrrolidin-1-yl)pyridine

To a cooled (0° C.) solution of(R)-(2-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)methanol (0.081 g, 0.38mmol) in dichloromethane was added SOCl₂ (0.450 g, 3.8 mmol) and thereaction mixture was allowed to warm to ambient temperature. After 1 h,the reaction mixture was concentrated and azeotroped with toluene toprovide (R)-3-(chloromethyl)-2-(3-fluoropyrrolidin-1-yl)pyridine (0.080g, 92%) as a clear oil. MS (ESI) m/z 215 [M+H]⁺.

Step 4:(R)-2-((2-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)methoxy)-6-hydroxybenzaldehyde

To a solution of(R)-3-(chloromethyl)-2-(3-fluoropyrrolidin-1-yl)pyridine (0.080 g, 0.35mmol) and 2,6-dihydroxybenzaldehyde (0.130 g, 0.94 mmol) in DMF wasadded potassium carbonate (0.190 g, 1.4 mmol) and the reaction mixturewas heated (60° C.). After 30 minutes, the DMF was removed and theresulting residue was reconstituted in CH₂Cl₂ and filtered through aplug of silica (EtOAc/hexanes, 1:1). Purification Prep-HPLC provided(R)-2-((2-(3-fluoropyrrolidin-1-yl)pyridin-3-yl)methoxy)-6-hydroxybenzaldehyde(8 mg, 5% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 11.71 (dd, J=8.4, 0.7 Hz,1H), 10.21 (d, J=0.5 Hz, 1H), 8.10 (dd, J=4.8, 1.9 Hz, 1H), 7.71 (dd,J=7.4, 1.9 Hz, 1H), 7.52 (t, J=8.4 Hz, 1H), 6.73 (dd, J=8.6, 0.7 Hz,1H), 6.71 (dd, J=7.4, 5.0 Hz, 1H), 6.53 (dt, J=8.4, 0.7 Hz, 1H), 5.40(dd, J=54.2, 3.3 Hz, 1H), 5.28 (d, J=11.3 Hz, 1H), 5.17 (d, J=12.0 Hz,1H), 3.91-3.56 (m, 4H), 2.21-1.93 (m, 2H); MS (ESI) m/z 317 [M+H]⁺.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

Throughout the description of this invention, reference is made tovarious patent applications and publications, each of which are hereinincorporated by reference in their entirety.

The invention claimed is:
 1. A compound selected from the groupconsisting of

or an N oxide thereof, wherein, R¹⁴ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl,COR¹⁵ or COOR¹⁵; R¹⁵ is optionally substituted C₁-C₆ alkyl, optionallysubstituted C₆-C₁₀ aryl, optionally substituted 5-10 membered heteroarylcontaining up to 5 ring heteroatoms, or optionally substituted 4-10membered heterocycle containing up to 5 ring heteroatoms, wherein theheteroatom is selected from the group consisting of O, N, S, andoxidized forms of N and S; x is 0, 1, or 2; and m is 0, 1 or
 2. 2. Acomposition comprising a compound of claim 1 and at least onepharmaceutically acceptable excipient.
 3. A method for increasing oxygenaffinity of hemoglobin S in a subject, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of claim 1.